Bioactive nanofibers are a new and fast-growing study field that has a high application potential in biomedical fields such as tissue engineering, drug and gene release and biosensor applications. The studies in the biomaterials field have been considerably advanced by using biocompatible and biodegradable materials with materials having bioactivity. Bioactivity becomes useful especially in tissue engineering studies [Wisse, E. Spiering, A. J. H., Dankers, P. Y. W., Mezari, B., Magusin, P. C. M. M., Meijer, E. M., J. Of Pol. Sci. Part A:Pol. Chem. 1764-1771, (2011)].
Tissue or organ loss is a significant health problem and there are lots of problems experienced in traditional treatment methods. The object of the tissue engineering studies; is to enable fixing, replacing or improving the function of certain tissues and organs by overcoming the limitations experienced in the traditional methods. The tissue engineering studies that comprise implementation of a functional, natural, synthetic or semi-synthetic tissue or organ imitation create an alternative or complementary solution potential for traditional methods [Tian, F., Tao, N. H., Tong, T., Gai, W. X., Applications of electro spun nanofibers, Chinese Science Bulletin, 53, 15, 2265-2286, (2008)].
In tissue engineering studies the cells are in vitro seeded on a scaffold. The cells proliferate on the scaffold, migrate and differentiate to specific tissues by secreting extracellular matrix (ECM) components required for formation of the tissue [Sachlos, E., Czernuszka, J. T., Making Tissue Engineering Scaffolds Work. Review On The Application Of Solid Freeform Fabrication Technology To The Production Of Tissue Engineering Scaffolds, European Cell And Materials, 5, 29-40, (2003)]. For tissue engineering to be successful, the scaffold that will provide structural support to the cells and cell-matrix (scaffold) interactions that will govern the tissue growth shall be emphasized. The scaffold structure plays a key role in tissue engineering since it mimics the natural ECM structure. The scaffold that serves as the temporary support during the time until the cells from natural ECM provides chemical, morphologic and structural signals for formation of the targeted tissue [Zhang, X., Reagan, M. R., Kaplan, D. L., Electro spun silk biomaterial scaffolds for regenerative medicine, Advanced Drug Delivery Reviews, 61, 988-1006, (2009)].
In tissue engineering, the material that will be used in scaffold design must be biocompatible, biodegradable and in a porous structure having a high surface area [Spagnuolo, M., Karpuz, O., Liu, L., Fabrication and Degradation of Electro spun Scaffolds from L-tyrosine Based Polyurethane Blends for Tissue Engineering Applications, Journal of Nanotechnology, (2011)]. Nanofibers have a high specific area and excellent pore connections due to their small fiber diameters. The scaffolds formed by nanofibers are ideal structures for being used in tissue engineering applications since they mimic ECM fibril structure and since they provide signals that stimulate cellular organization, vitality and function [Nisbet, D. R., Forsythe, J. S., Review Paper: A Review Of The Cellular Response On Electro spun Nanofibers For Tissue Engineering, Journal Of Biomaterials Applications, 24, 7-29, (2009)].
A bioactive molecular cell migration such as a specific peptide or growth factor may stimulate processes such as growth or differentiation. Therefore, it is possible to obtain bioactive nanofibers by binding bioactive molecules to nanofiber mats obtained through electrospinning method. Electrospinning method is particularly suitable for obtaining bioactive polymer nanofibers. This method is simple and inexpensive for large scale production. In addition, nanofibers provide a larger surface area and thus more protein loading capacity when compared to protein immobilized polymer film surfaces. Bringing bioactivity to polymers is performed by mixing the bioactive molecules and polymer solutions or by covalently binding the biomolecule to the polymer via functional groups. However, mixing the bioactive molecule with the polymer solution is not an efficient method since it does not allow controlling the activity of the molecule and it does not allow the molecule to bind to the desired region. In this situation, a covalent bond is advantageous [Wisse, E. Spiering, A. J. H., Dankers, P. Y. W., Mezari, B., Magusin, P. C. M. M., Meijer, E. M., J. Of Pol. Sci. Part A:Pol. Chem. 1764-1771, (2011)].
In making the polymer nanofibers functional by using covalent binding with proteins, usually the existence of carboxylic acid groups in the polymer structure is utilized. For this purpose, water soluble agents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) are commonly used. EDC that has low toxicity enables the formation of amide bonds between the carboxylic acid groups and amino groups. They are used in the covalent binding of biomolecules on polymers by being used together with NHS [Zheng, W., Zhang W., Jiang, X., Biometic Collagen Nanofibrous Materials for Bone Tissue Engineering, ADVANCED ENGINEERING MATERIALS, 12, 9, B451-B466, (2010)].
Nanofibers are obtained from various different polymers as synthetic or natural. In tissue engineering studies, the nanofiber structures that are obtained by electrospinning method from poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL) and poly(α-hydroxyl acid) group polymers that are copolymers thereof are frequently used in making scaffolds [Yow, S. Z., Lim, T. H., Yim, E. K. F., Lim, C. T., Leong, K. W., A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering, Polymers, 3, 527-544, (2011)].
Using conductive polymers in tissue engineering studies stands as a new approach [Li, M., Guo, Y., Wei, Y., MacDiarmid, A. G., Lelkes, P. I., Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications, Biomaterials, 27, 2705-2715, (2006)]. Conductive polymers have the potential of governing the adhesion, migration, protein secretion and DNA synthesis functions of cells that respond to electrical stimulation such as nerve, bone, muscle and cardiac cells. The conductivity of the conductive polymers that are a special class of materials having electronic and ionic conductivity is originating from the conjugated double bonds in the polymer backbone [Ravichandran, R., Sundarrajan, S., Venugopal, J. R., Mukherjee, S., Ramakrishna, S., Applications of conducting polymers and their issues in biomedical engineering, J. R. Soc. Interface, 7, S559-S579, (2010)]. Using pyrrol, thiophene and aniline derived from the heteroaromatic monomers and conductive polymers which are derivatives thereof gains a substantial interest in tissue engineering. However, since said biocompatible and conductive polymers do not have regions on their surfaces that help cell recognition, it limits the use of nanofiber mats obtained from said polymers in various biomedical fields such as tissue engineering applications.
Therefore, in order to increase biocompatibility and make nanofibers gain biofunctionality, bioactive molecules can be included into the nanofiber scaffold. It is known that including RGD peptide and various growth factors into the nanofiber mats increases the cell behavior and adhesion in tissue engineering scaffolds [Plessis, D. M, Fabrication and characterization of anti-microbial and biofouling resistant nanofibers with silver nanoparticles and immobilized enzymes for application in water filtration, (Master's Thesis), University of Stellenbosch, Faculty of Science Department of Biochemistry, (2011)]. Ravichandran et al. (2010) covalently bound the RGD peptide (Arg-Gly-Asp) to the polypyrol layer obtained by electropolymerization and enabled use of the polymer in orthopedic applications as a bioactive material (Ravichandran, R., Sundarrajan, S., Venugopal, J. R., Mukherjee, S., Ramakrishna, S., Applications of conducting polymers and their issues in biomedical engineering, J. R. Soc. Interface, 7, S559-S579, (2010)]. The carboxyl groups (—COOH) in the aspartic acid (Asp) amino acid that is present in the RGD peptide structure have the ability to make a covalent bond with a polymer having chemically active parts.
In the invention, production and detailed characterization of conductive and bioactive nanofiber mats that have a high potential of use in tissue engineering field is realized. Accordingly, in situ polymerization of the poly(m-anthranilic acid) (P3ANA) conductive polymer in polycaprolactone (PCL) solution which is a biocompatible polymer is realized. Nanofiber mats are produced by electrospinning method from the obtained polymer solution. P3ANA contains carboxyl group in the aniline backbone and has a great potential of use due to its processability in aqueous, non-aqueous and polar solvents [Avci, Z. M., Sarac, A. S., Transparent Poly(methyl methacrylate-co-butyl acrylate) Nanofibers, J. Appl. Polym. Sci. (2013) DOI: 10.1002/APP.39705]. By utilizing the presence of —COOH group in the P3ANA structure, the RGD peptide (Arg-Gly-Asp) that is a cell adhesion protein will covalently bind to the nanofiber mat by using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) agents and the mat is made bioactive.